The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer.

Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood.

Studying Neuronal Function Ex Vivo Using Optogenetic Stimulation and Patch Clamp.

Optogenetics has become a key method to interrogate the function of neural populations and circuits in the brain. This technique combines the targeted expression of light-activated proteins with subsequent manipulation of neural activity by light. Opsins such as Channelrhodopsin-2 (ChR2), which is a light-gated cation-channel, can be fused to or coexpressed with fluorescent proteins to allow for visualization and concurrent activation of neurons and their axonal projections.

Evaluation of Lipid In-Source Fragmentation on Different Orbitrap-based Mass Spectrometers.

Natural lipidomes represent a complex mixture of lipid molecular species with a variety of biological and signaling functions. Modern mass spectrometry (MS)-based analytical platforms are often used to resolve the complexity of natural lipidomes. The quantitative transfer of lipid molecular species in the gas phase during the electrospray ionization required for MS analysis might be challenged by lipid in-source fragmentation (ISF) hampering their accurate identification and quantification.

Mapping Unsaturation in Human Plasma Lipids by Data-Independent Ozone-Induced Dissociation.

Over 1500 different lipids have been reported in human plasma at the sum composition level. Yet the number of unique lipids present is surely higher, once isomeric contributions from double bond location(s) and fatty acyl regiochemistry are considered. In order to resolve this ambiguity, herein, we describe the incorporation of ozone-induced dissociation (OzID) into data-independent shotgun lipidomics workflows on a high-resolution hybrid quadrupole-Orbitrap platform.

Analysis of oxidised and glycated aminophospholipids: Complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry.

The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules regulating cell death.

Rational selection of reverse phase columns for high throughput LC-MS lipidomics.

Natural lipidomes are characterized by extremely high complexity and dynamic range of lipid concentrations. Furthermore, high diversity of lipid physicochemical properties requires high resolving powers for both chromatographic and mass spectrometric analytical platforms. Reverse-phase chromatography coupled with data-dependent MS/MS acquisition is one of the most popular techniques in untargeted lipidomics.

A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies.

Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples.

An endogenous bile acid and dietary sucrose from skin secretions of alkaloid-sequestering poison frogs.

The skins of Madagascar poison frogs (Mantella) and certain Neotropical poison frogs (Epipedobates, Dendrobates) secrete the new bile acid tauromantellic acid (1), which was found in both wild-caught and captive-born frogs. This is the first molecule of endogenous origin detected in skin secretions from these taxa. Sucrose was also detected in secretions from wild-caught Mantella but not in captive-born frogs, suggesting a dietary origin.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis.

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors.

Kasstasin: A novel potent vasoconstrictor peptide from the skin secretion of the African red-legged running frog, Kassina maculata.

Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated.

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos.

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.

Kassorins: novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis.

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.

SLoMo: automated site localization of modifications from ETD/ECD mass spectra.

Recently, software has become available to automate localization of phosphorylation sites from CID data and to assign associated confidence scores. We present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to ETD/ECD mass spectra. Furthermore, SLoMo caters for both high and low resolution data and allows for site-localization of any UniMod post-translational modification.

Multicomponent reactions in fungicide research: the discovery of mandipropamid.

Isocyanide-based multicomponent reactions of the Ugi- and Passerini-type have been valuable tools for the rapid exploration of the novel fungicidal compound classes of phenylglycinamides and mandelamides. Mandipropamid (6), which was discovered during this derivatisation, displays excellent activity against the economically important phytopathogens Phytophthora infestans (potato and tomato late blight) and Plasmopara viticola (grape downy mildew).

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased.

Synthesis and fungicidal activity of N-2-(3-methoxy-4-propargyloxy)phenethyl amides. Part II: anti-oomycetic mandelamides.

Novel types of anti-oomycetic compounds have been designed and prepared. The synthetic approach to these mandelamides is outlined. Biological data demonstrate their high efficacy against important plant diseases such as tomato and potato late blight (Phytophthora infestans De Bary) and grape downy mildew (Plasmopara viticola Berliner & de Toni).

Data-dependent electron capture dissociation FT-ICR mass spectrometry for proteomic analyses.

Electron capture dissociation (ECD) offers many benefits for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Recent developments have reduced the time scale required for ECD to milliseconds resulting in the technique's compatibility with on-line separation techniques, e.g.

Production and properties of nanoelectrospray emitters used in Fourier transform ion cyclotron resonance mass spectrometry: implications for determination of association constants for noncovalent complexes.

Electrospray ionization (ESI) is extensively used in the analysis of biological compounds; yet some fundamental properties of this technique are not completely understood. It is widely recognized that care should be exercised when noncovalent complexes are being studied by ESI, since weak noncovalent binding can be broken or formed during the desolvation process. In the present work, spectra from the noncovalent complex, vancomycin/diacetyl-L-lysyl-D-alanyl-D-alanine, obtained from ESI and from nanoelectrospray ionization (nanoESI), have been compared.

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites.

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins.

A rapid nonradioactive peptide phosphorylation assay.

Radioactive assays are commonly employed to monitor protein or peptide phosphorylation. They not only have all the disadvantages related to radioactivity, but also require large amounts of sample. An alternative is the use of mass spectrometric peptide mapping with sensitivities in the fmole range.

Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain.

A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect.

Use of an acid-labile surfactant as an SDS substitute for gel electrophoresis and proteomic analysis.

Modern protein identification and analysis relies largely on proteolytic in-gel digestion of proteins separated during polyacrylamide gel electrophoresis (PAGE) followed by mass spectrometric (MS) measurement of the extracted peptides. Sodium dodecyl sulfate (SDS) is routinely used in nonnative PAGE. However, SDS can interfere with MS.